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The slice spectrum at 1543 cm-1 is shown in Figure 11.14(II). A couple of cross peaks are developed at (1671, 1683), (1653, 1683), (1623, 1671), and (1623, 1653 cm-1) in the Amide I region of the asynchronous spectrum. The signs of the cross peaks suggest the following sequence of the H/D exchanges in the amide protons.
P-turns (1683 cm-1) > aggregated P-strands (1623 cm-1) > P-turns (1671cm-1) > a-helices (1653 cm-1)
Therefore, it seems that in the second time domain of the exchange, the amide protons in another kind of P-turns (1671 cm-1) are involved in the H/D exchanges. They are more accessible to the D2O molecules than the accessible parts of a-helix structures. Similarly, from the 2D correlation spectra of HSA, constructed from the nine spectra measured between 25.2 and 181 min after the beginning of H/D exchange process (Group III), Wu et al. concluded following sequence of spectral changes:
P-turns (1663cm-1 > P-turns (1683cm-1), random coil (1643,1540cm-1)
> a-helices (1653,1549cm-1) > aggregated P-strand (1623cm-1).
This study has demonstrated that 2D IR correlation spectroscopy study of the H/D exchanges in protein is a powerful tool to explore the kinetics of the H/D exchanges in HSA and to unravel poorly resolved Amide I, II, and IF regions. Asynchronous spectra are particularly useful to deconvolute Amide I, II, and IF regions and to provide the order in the H/D exchanges. The combination of PCA and 2D correlation analysis turns out to be very efficient in distinguishing the fast kinetics from the slow ones during the H/D exchange process.
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2D Correlation Spectroscopy and Chemical Reactions
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12 Protein Research by Two-dimensional Correlation Spectroscopy
2D correlation spectroscopy is a popular tool in the analysis of IR spectra of proteins.1-22 For example, it enables the highly overlapped Amide I, II, and III bands of proteins to be resolved into component bands ascribed to various secondary structures. It is also possible to investigate the correlations among Amide I, II, and III bands. For example, Murayama et al. demonstrated that 2D correlation spectroscopy is also useful for exploring intensity variations of COOH bands of glutamic and aspartic acid residues of proteins.18 Wang et al. applied 2D NIR correlation spectroscopy to monitor protein-water interaction.14 One of the earlier examples of the application of 2D IR correlation spectroscopy to protein research was concerned with the secondary structure of myoglobin.6 Hydrogen-deuterium (H/D) exchange of the amide protons was employed as an external perturbation to generate the 2D spectra. Since that time 2D IR correlation spectroscopy has been used extensively to explore the secondary structure of proteins. Sefara et al. studied thermal transitions in -lactoglobulin (BLG) using both 2D IR and 2D NIR spectroscopy.7 8 Smeller et al.9,10 and Dzwolak et al.11 reported 2D IR studies of pressure-dependent structural modifications of proteins. Schultz et al. carried out 2D IR, 2D NIR, and 2D IR-NIR heterospec-tral correlation analysis of the thermal unfolding of ribonuclease A (RNase A).12 It was reported that different structural elements in RNase A respond slightly differently to a temperature increase. Fabian et al. also reported 2D IR correlation spectroscopy study on noncooperative unfolding of the Cro-V55C repressor protein.13 They pointed out that in combination with protein engineering, the 2D IR approach provides insight into the impact of point mutations on the stability of proteins and the presence of folding intermediates as predicted from molecular modeling. Wang et al. reported 2D NIR correlation analysis of concentration-dependent spectral changes in ovalbumin solutions at various temperatures.14 New insight has been gained into the hydration as well as unfolding process of secondary structures of ovalbumin by studying temperature-dependent correlation patterns in 2D NIR spectra. Wu et al. used slice spectra of 2D NIR maps generated from the concentration-perturbed spectral variations of HSA solutions